Xiao Annie, Gibbons Anne E, Luker Kathryn E, Luker Gary D
Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, MI, USA.
Departments of Biomedical Engineering and Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA.
Tomography. 2015 Dec;1(2):115-124. doi: 10.18383/j.tom.2015.00163.
Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time, to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from two-dimensional cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity.
基因编码的荧光共振能量转移（FRET）报告分子是在标准二维细胞培养中以单细胞分辨率分析细胞信号传导和功能的强大工具，但这些报告分子很少应用于三维环境。供体和受体分子之间的FRET相互作用通常由相对荧光强度的变化来确定，但光吸收的波长依赖性差异使这种分析方法在三维环境中变得复杂。在这里，我们报告了带有相量分析的荧光寿命成像显微镜（FLIM），这是一种实时逐像素显示荧光寿命的方法，用于量化稳定表达基因编码FRET报告分子的乳腺癌细胞中的凋亡。这种显微成像技术使我们能够在从二维细胞培养、含有癌细胞和骨髓基质细胞的球体以及患有原位人乳腺癌异种移植的活体小鼠等不同环境中识别单个乳腺癌细胞中治疗诱导的凋亡。使用这种成像策略，我们表明联合靶向糖酵解和谷氨酰胺途径的代谢疗法显著降低了整体乳腺癌代谢并诱导了凋亡。我们还确定骨髓基质细胞的不同亚群控制着乳腺癌细胞对化疗的耐药性，这表明不同骨髓微环境中恶性细胞的治疗反应存在异质性。总体而言，本研究确立了带有相量分析的FLIM作为基于细胞的实验和活体小鼠中凋亡的成像工具，能够对治疗效果和异质性进行实时、细胞水平的评估。
